This project will continue our evaluations of falcipain cysteine proteases as antimalarial drug targets. Recent studies indicate that falcipain activities are required through the erythrocytic life cycle of malaria parasites, and thus that these enzymes are promising drug targets. Indeed, cysteine protease inhibitors block hemoglobin hydrolysis, prevent the development of intraerythrocytic parasites, inhibit erythrocyte rupture and invasion, and cure malaria-infected mice. Efforts to develop falcipain inhibitors as antimalarial drugs are ongoing, and evaluations of the antimalarial effects of new inhibitors will make up an important part of this project. In addition, we will carry out studies designed to clarify the optimal therapy directed against plasmodial cysteine proteases, including the most appropriate class of compound, specificity of action, and type and timing of activity. We will also evaluate the importance of the reversibility of action of inhibitors and ease of resistance selection, and we will evaluate the efficacy and effects on resistance selection of combination antimalarial therapy. We hypothesize that falcipain inhibitors will prove to be effective antimalarials, but that optimal use of these compounds must consider the appropriate class of compound, best-suited mechanism and timing of inhibition, the specific inhibition of different falcipains, the ease of selection of drug-resistant parasites, and the utility of combination with other classes of antimalarials. We further hypothesize that certain combinations of cysteine protease inhibitors and other agents will provide synergistic antimalarial activity and prevent the development of antimalarial drug resistance, Collaborations are in place to evaluate multiple classes of cysteine protease inhibitors as antimalarial agents. The specific aims of the project will be: 1) to evaluate the antimalarial effects of falcipain inhibitors synthesized by our collaborators, 2) to heterologously express falcipains and homologs from other plasmodia, 3) to select and characterize malaria parasites resistant to cysteine protease inhibitors, and 4) to evaluate the efficacy and impact upon drug resistance of combinations of cysteine protease inhibitors and other agents. The studies proposed here will allow us to identify and better characterize optimal cysteine protease inhibitor antimalarial compounds, and continue progress towards the development of new falcipain inhibitors as antimalarial drugs.